Process for sealing microplates utilizing a thin polymeric film

ABSTRACT

A sealing member for a multi-well microfiltration plate comprises a flexible sealing material which, in response to the application of differential pressure, will flex or collapse in the direction of the filtration along the contour of each individual well. A process maintains a differential pressure substantially constant over a multi-well microfiltration plate comprising placing said flexible sealing member over that surface of the plate having well openings, stretching the flexible sealing member so as to seal the perimeter of each individual well, creating a differential pressure around said plate covered with said seal and filtering a media from each occupied well at a rate independent of the filtration rate of any other well while maintaining a substantially constant differential pressure around the plate until filtration in the last well containing media is complete.

BACKGROUND OF THE INVENTION

The invention is directed to the use of a thin, pliable film used forsealing the cell(s) of a microplate. More specifically, the invention isdirected to an economical, simple and effective means of sealing avacuum dawn or pressure forced microfilter plate which seals the cellsthereof individually, thus allowing the filtration in each cell toproceed undisturbed by the status of filtration in other cells. Thesubject sealing means or system ensures that the vacuum or pressurelevel above or below the plate is not affected by completion of thefiltration in one or more cells while other cells remain in thefiltering status.

Microfiltration plates are fitted with membrane or depth filters and areintended to allow for the micro or ultrafiltration of multiple samplessimultaneously. Generally, a vacuum is pulled under the plate, orpressure is applied over the plate, as a driving force for thefiltration to be completed. An inherent problem with these devices isthat not all wells within a single microplate, which may contain as manyas 96 or more wells, filter at the same rate. This difference infiltration rate among wells of the same plate may be due to variousparameters of the filtration set-up, such as the use of a defectivemembrane or other filter media in a particular well(s), a defective sealon the well(s), sample variances, and other differences in theindividual wells or samples. Once all of the sample liquid has beenremoved from a well, air replacing the liquid and flowing through thefiltration well alters the vacuum or pressure driving force retardingthe rate of filtration in the remaining wells which continue to functionor filter. Air breakthrough may also cause turbulence below the filter,enhancing the possibility of splashing and cross-contamination of liquidin the receiver reservoir(s).

Attempts to solve the foregoing problem have been varied. Many involvethe use of different seal designs. Among them are the use of perforatedpolyester or cellophane tapes, and the use of a rigid or semi-rigid capwhich fits the top of the plate. The main drawback to these types ofseals is the rigidity thereof.

U.S. Pat. Nos. 5,464,541 and 5,462,878 describe prior art rigid seals.These devices prevent air breakthrough. However, they create anotherproblem. As the liquid filters under the seals, a vacuum is createdabove the liquid. Every time the air gap above the filtering liquiddoubles, the pressure driving force for filtration halves, causing anever decreasing filtration rate. This effect is dramatically magnifiedas the mount of liquid placed in each well is increased. An alternativeto a rigid cap seal on the microfiltration device is to individuallyseal each well as the liquid volume approaches the filter, a scenariowhich requires constant attention.

Therefore, if no sealing device is used, filtration efficiency of theentire microplate decreases rapidly once filtration of a first well orcell is completed and air breakthrough occurs. If a rigid sealing deviceis used to prevent air breakthrough, filtration efficiency of each welldecreases at different rates, depending on the amount of liquidoriginally placed in each well and its filtration rate.

Other attempts to correct the seal problem described above include theuse of a compliant, reusable sheet which does not seal the wells, butrather creates a bubble-point as all of the sample is drawn from a well,stopping filtration in that well. In this device, embodied in U.S. Pat.No. 4,927,604, no seal is created within the meaning of the currentinvention.

The use of a pliable polymeric film for sealing microplates solves boththe air breakthrough and decreasing filtration rate problems discussedabove.

It is therefore an object of the subject invention to provide a meansfor sealing a microfiltration device such that filtration of all wellstherein proceeds to completion effectively and efficiently.

It is yet another object of the invention to provide a means for sealingindividual wells within a multi-well filtration plate, as each wellempties, so as not to disturb the filtration process taking place in theremaining wells.

It is another object of the invention to use a pliable polymeric film toseal the microplate as a whole and each well individually and preventdisturbance of the filtration process overall by collapse of the filminto each well, at the particular filtration rate within each well,following the reduction in liquid level in the well, as the filtrationprocess proceeds.

SUMMARY OF THE INVENTION

The invention relates to a sealing member for a multi-wellmicrofiltration plate comprising a flexible sealing material which, inresponse to the application of differential pressure, will flex orcollapse in the direction of the filtration along the contour of eachindividual well. The invention further relates to a process formaintaining a differential pressure substantially constant over amulti-well microfiltration plate comprising placing said flexiblesealing member over that surface of the plate having well openings,stretching the flexible sealing member so as to seal the perimeter ofeach individual well, creating a differential pressure around said platecovered with said seal and filtering a media from each occupied well ata rate independent of the filtration rate of any other well whilemaintaining a substantially constant differential pressure around theplate until filtration in the last well containing media is complete.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plan view of a microfiltration plate;

FIG. 2 is a cross-sectional view along lines A--A of the microfiltrationplate shown in FIG. 1;

FIG. 3 is a cross-sectional view of a microfiltration plate with theinventive sealing cover in place, prior to use; and

FIG. 4 is a cross-sectional view of a microfiltration plate with theinventive sealing cover during use in a filtration process.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the use of a flexible seal to maintain theconstant pressure driving force on a microfiltration or ultrafiltrationplate. As the filtration process proceeds and the liquid level of eachcell or well decreases, the flexible seal described herein depressesinto the well or cell, following the liquid level. Thus, a vacuum isnever created over the liquid and the driving force is never decreasedor compromised, other than the force required to stretch the seal whichis negligible. Filtration in all wells or cells is allowed to proceedindividually, at its own rate, while a seal is maintained over eachindividual well or cell. Also, due to the individual sealing of eachwell, air breakthrough never occurs.

As used herein, the terms well and cell interchangeably refer to theindividual filtration chambers of a multi-chamber micro- orultra-filtration plate. Also, the term "microplate" as used hereinrefers to any multi-well filtration device, whether microfiltration,ultrafiltration, or other. Finally, the subject invention is equallyapplicable and well-suited for use in those instances where vacuum isdrawn from below the plate to cause the filtration to occur, or pressureis applied above the plate to force the filtration process. This isintended to further include any device in which a centrifuge of or anyother centrifugal device is used to effect filtration. In all instances,a pressure differential is being created across the film or the plate.Therefore, hereinafter, unless otherwise specified, use of the term"pressure differential" may refer to any of the above processes.

FIG. 1 is a plan view of a microfiltration plate 10, in this instancehaving ninety-six (96) wells 12 for filtering material. As is generallydone, the wells are identified by rows and column for ease in sampleidentification.

FIGS. 2-4 are a cross-sectional view along lines A--A of FIG. 1. In FIG.2, the wells 12 do not contain a material to be filtered. In FIG. 3, themicrofiltration plate 10 is covered with a sealing member 20 in keepingwith the invention. The wells 12 in FIG. 3 are shown containing amaterial to be filtered 22. FIG. 4 shows the microfiltration plate 10 ofFIG. 3, bearing sealing member 20, during a filtration process. Sealingmember 20 is shown to be collapsed in correlation to the filtration rateof each individual well 12, i.e., the sealing member 20 is collapsed toa different extent in each well in relation to the amount of material 22remaining in the well. It is further noted that sealing member 20 is insealing contact with the upper rim 14 of each well 12 during thefiltration process to prevent air break-through.

Any number of known flexible materials may be used to create theflexible seal which is the subject hereof. In selecting an appropriateflexible seal material, it is important to ensure compatibility of theflexible seal material with the material/media being filtered. Further,the ability of the flexible material to stretch easily and be easilyused with wells of varying sizes from plate to plate is important. Ifthe material used to form the subject sealing means returns to itsoriginal shape upon relaxation of the filtration driving force, whetherby employing a chemical treatment of the material or by employing aphysical treatment of the material, such as heating, it may beappropriately cleaned and reused. Materials that do not or cannot becaused to return to their original shape may be used in disposableapplications or scenarios. Suitable materials which meet the foregoingrequirements of pliability, such as any natural or synthetic rubber,include latex, silicone rubber, and other elastomeric materials withsimilar properties, and further include other plasticized orunplasticized polymers such as polyvinylidine chloride, polyvinylchloride, polyethylene, paraffin films, other suitable materials orblends thereof.

The sealing material may be affixed in a rigid perimeter frame devicefor ease in securing the sealing member to the microfiltration plate. Inthis embodiment, the sealing member would cover the tops of the wells ofthe plate and the frame would merely support the sealing member,allowing it to lay flat over the open ends of the wells and create aseal on each when a pressure differential is applied. The frame couldoptionally be used to effect an additional seal around the perimeter ofthe plate. This would hold the sealing means in constant position withrespect to the plate as a whole, allowing for an individual seal to beachieved over each well opening.

Use of the subject seal provides yet another benefit in the area ofmicrofiltration. Often times microfiltration plates are used in thebiomedical sciences for testing highly sensitive samples, not only fromthe standpoint of the sample itself, but also from the standpoint ofoperator safety. Use of the subject sealing member affords maintenanceof the sample in a non-contaminated state. Further, because air flowthrough the wells upon removal or filtration of the liquid sample doesnot occur, prior problems caused by the sample drying out are foregone.

EXAMPLES Example I

Test solutions were pipetted into the individual wells of a Uniplateassembly. While it is not necessary to the proper function of theinvention that all wells be filled, in this instance all were filled. Avacuum was applied to achieve filtration of the sample within the wells.A robber latex sealing member, 0.010 inches thick, was then placedacross the assembly. It was ascertained that all edges around each wellwere sealed. As filtration proceeded the process in all wells did notproceed at the same filtration rate. In those wells where the rate wasfaster, the rubber latex sealing member was observed to collapse intothe well, individually, at the same rate at which the liquid was beingfiltered out of the well. No air breakthrough or other disturbance ofthe filtration rate/process in the individual wells was observed.

Example II

In this Example II, the Uniplate assembly was prepared just as inExample I with the exception that the sealing member was placed on theassembly prior to applying the vacuum. Filtration proceeded just as inExample I, with the filtration rate varying from well to well. Therubber latex sealing member was once again observed to collapse intoeach well individually at the same rate at which the liquid was filteredfrom the well. There was no breakthrough or other disturbance of thefiltration rate/process in the wells.

As was stated hereinabove, the invention described herein is not onlysimple and cost effective, but it is also highly efficient and affordsthe operator greater freedom to monitor other aspects of the filtrationprocess without the need to individually seal wells as they empty inorder to maintain the desired pressure differential across the plate.

We claim:
 1. A process for maintaining a differential pressuresubstantially constant over a multi-well microfiltration device, saiddevice comprising an upper surface having multiple well openings, eachof said well openings having a perimeter on the upper surface of saiddevice and a central opening for receiving media to be filtered,sidewalls extending downward from said perimeter below said centralopening, and a bottom surface such that a well for retaining a media tobe filtered is formed, said process comprising:occupying at least twowells of said device with a media to be filtered; creating adifferential pressure around said device; placing a flexible sealingmember over said upper surface and each of said multiple well openingsof said device; stretching said flexible sealing member so as to sealsaid perimeter and said central opening of each individual well of saidmulti-well micro filtration device; and filtering said media from eachoccupied well of said device at a rate independent of the filtrationrate of any other well of said device, while maintaining a substantiallyconstant differential pressure around said device such that saidflexible sealing member collapses into each well of said device,preventing air breakthrough, at a rate which varies among said wells ofsaid device correspondingly to said independent rate of filtration ofeach occupied well, until filtration in all wells containing media iscomplete.
 2. The process of claim 1 wherein said sealing membercomprises a flexible material selected from the group consisting ofnatural rubbers, synthetic rubbers, and plasticized and unplasticizedpolymeric materials.
 3. The process of claim 1 wherein said sealingmember comprises a flexible material selected from the group consistingof latex, silicon rubber polyvinylidine chloride, polyvinyl chloride,polyethylene, paraffin films, and combinations thereof.
 4. The processof claim 1 wherein said sealing member further includes a rigid frameinto which said sealing member is affixed, said frame corresponding insize and shape to said upper surface of said multi-well microfiltrationdevice and being fixable thereto.
 5. The process of claim 1 wherein saiddifferential pressure is created by drawing a vacuum on said device. 6.The process of claim 1 wherein said differential pressure is created bythe application of pressure to the atmosphere above said wells of saiddevice.
 7. The process of claim 1 wherein said differential pressure iscaused by centrifugal action used to affect filtration.
 8. A process formaintaining the uncontaminated nature of samples to be filtered orrecovered from individual wells of a multi-well microfiltration device,each said well having an upper perimeter and a central opening withinsaid upper perimeter for receiving sample to be filtered, said processcomprising placing said samples in various wells of a multi-wellmicrofiltration device, applying a differential pressure, and sealingsaid wells with a single flexible sealing member which collapses intoeach well at a rate of collapse independent of the rate of collapse ofsaid sealing member into each other well and consistent with the rate offiltration within each well, such that each well remains entirely sealedthrough a filtration process and said samples cannot be contacted byagents outside said wells and said samples cannot contact agents outsidesaid wells.